The cornified envelope is a fifteen (15) nm thick insoluble protein layer that is formed under the plasma membrane in the upper layers of epidermis and keratinizing stratified epithelium (Reichert, U. et al. (1993) Molecular Biology of the Skin, 107-150). It appears to play a major role in the physical barrier properties of the stratum comeum (Elias, P. M. and D. S. Friend (1975) J. Cell. Biol. 65:180-191). The envelope is formed from several precursor proteins by the calcium dependent enzyme transglutaminase, which catalyzes formation of xcex5-(xcex3-glutamyl) lysine crosslinks (Polakowska, R. R. and L. A. Goldsmith (1991) Physiology, Biochemistry and Molecular Biology of the Skin, 168-201) that are resistant to proteolytic digestion. It has been postulated that crosslinking of an envelope related protein, involucrin, to the plasma membrane is a first step in envelope assembly (Ishida-Yamamoto, A. et al. (1997) J. Invest. Dermatol. 108:12-16). This is followed by crosslinking of the less abundant precursors such as SPRR proteins, elafin, envoplakin, filaggrin, keratin filaments and cystatin a (Steinert, P. M. and L. N. Marekov (1995) J. Biol. Chem. 270:17702-17711; Takahashi, H. et al. (1997) J. Invest. Dermatol. 108:843-847; Ruhrberg, C. et al. (1996) J. Cell. Biol. 134:715-729; Takahashi, M. et al. (1996) Arch. Biochem. and Biophys. 329:123-126). Finally loricrin covers the cytoplasmic side of the envelope (Candi, E. et al. (1995) J. Biol. Chem.270: 26382-26390).
Several reports have suggested that multiple components are necessary for envelope structure and function. Involucrin for example acts as the framework for the attachment of other envelope components and is covalently linked to the lipids which are important components of the barrier of stratum comeum cells (Downing, D. T. (1992) J. Lipid Res. 33:301-313). The pancornulin proteins have been shown to act as molecular bridges and are able to cross-link with two different proteins (Li, V. W. et al. (1996) Dermatology Clinics 745-751). Loricrin can impart flexibility as a result of its high glycine content and insolubility from disulfide bonds.
Gene mutation and knockout studies have been used to gather information on the function of epidermal proteins, with the keratins being the most well known (Fuchs, E. et al. (1992) PNAS 89:6906-6910; Vassar, R. et al. (1991) Cell 64:365-380). Only one study of envelope related proteins has been reported, a loricrin knockout mouse (deviragh, P. A. et al. (1996) J. Invest. Dermatol. 106:844; deviragh, P. A. et al. (1997) J. Invest. Dermatol. 108:555). Heterozygous mice are normal, while homozygotes have abnormal skin during the first few days, but the animals appear normal as adults However, the mice have a defect in barrier function and respond abnormally to the application of some chemicals. A mutation of the loricrin gene has been observed in patients with a rare autosomal dominant palmoplantar keratoderma, Vohwinkel""s Keratoderma, as well as in Progressive, Symmetric Erythrokeratoderma (Ishida-Yamamoto, A. et al. (1997) J. Invest. Dermatol. 108:12-16). Loss of epidermnal transglutaminase activity from mutations in the gene results in the human disease, lamellar ichthyosis, which is characterized by ia thickened stratum corneum, disturbed epidermal keratinization and inflammatory changes (Huber, M. et al. (1995) Science 267:525-528).
The present invention is based, in part, on the discovery of the gene which encodes the epidermal protein, sciellin. Accordingly, the present invention features a purified or isolated preparation or a recombinant preparation of sciellin, or a sciellin polypeptide.
In a preferred embodiment, sciellin has at least 60% to about 70%, more preferably at least about 80%, even more preferably at least about 90% to about 95%, and most preferably at least about 99% sequence identity with human sciellin, e.g., the human sciellin of SEQ ID NO:2. Sciellin can be identical to a human sciellin sequence, e.g., that of SEQ ID NO:2. In another embodiment, sciellin is encoded by a nucleic acid molecule which hybridizes under stringent conditions to a nucleic acid molecule of the nucleic acid sequence shown in SEQ ID NO:1. In addition, sciellin can have substantially the same electrophoretic mobility as human sciellin, e.g., it appears as an electrophoretic band of about 75.3 kDa on reducing gels. Yet another preferred embodiment of the invention features a sciellin which is reactive with a sciellin-specific antibody, e.g., an antibody which binds to the epitope recognized by mAb 34D11, or a polyclonal antibody SC4. Antibodies against sciellin can be made by methods exemplified herein.
In another preferred embodiment, sciellin is expressed by a recombinant cell, e.g., a bacterial cell, a cultured cell (e.g., a cultured eukaryotic cell) or a cell of a non-human transgenic animal. Cultured cells can include CHO cells or SF8 cells. Expression of sciellin in a transgenic animal can be general or can be under the control of a tissue specific promoter. Preferably, one or more sequences which encode sciellin or a fragment thereof are expressed in a preferred cell-type by a tissue specific prtomoter, e.g., a K14 promoter. Exemplary sequences encoding fragments of sciellin include, e.g., a sequence encoding the central domain of sciellin, e.g., one or more of repeats 1-16, or a sequence encoding a LIM domain.
In a preferred embodiment, the recombinant sciellin differs from sciellen isolated from tissue in one or more of the following: its pattern of glycosylation, myristilation, phosphorylation, or other posttranslational modifications.
In a preferred embodiment, the recombinant sciellin preparation is free of other keratinocyte proteins, placental proteins, or other human proteins.
In a preferred embodiment, the recombinant sciellin preparation contains at least 1, 10, or 100 xcexcg of sciellin, or a sciellin polypeptide.
In a preferred embodiment, the recombinant sciellin preparation contains at least 1, 10, or 100 mg of sciellin, or a sciellin polypeptide.
In a preferred embodiment, the sciellin polypeptide has the following biological acitivities: 1) it is a precursor of the cornified envelopelof keratinizing tissues; 2) it provides structural support to the comified envelopes of stratum corneum cells; 3) it promotes adhesion between tissue elements; 4) it promotes intracIellular signalling; 5) it defines cell shape; 6) it can act as an adaptor element to promote the assembly and targeting of multiprotein complexes; 7) it forms homotrimeric beta helices; (8) it is involved in the terminal differentiation of keratinocytes; and (9) it plays a role in development. In other preferred embodiments: the sciellin polypeptide includes an amino acid sequence with at least 60%, 80%, 90%, 95%, 98%, or 99% sequence identity to an amino acid sequence from SEQ ID NO:2; the sciellin polypeptide includes an amino acid sequence essentially the same as the amino acid sequence in SEQ ID NO:2; the sciellin polypeptide is at least 5, 10, 20, 50, 100, or 150 amino acids in length; the sciellin polypeptide includes at least 5, preferably at least 10, more preferably at least 20, most preferably at least 50, 100, or 150 contiguous amino acids from SEQ ID NO:2; the sciellin polypeptide is either, an agonist or an antagonist, of a biological activity of naturally occurring sciellin; the sciellin polypeptide is a vertebrate, e.g., a mammalian, e.g. a primate, e.g., a human, sciellin polypeptide.
In preferred embodiments: the sciellin polypeptide is encoded by the nucleic acid in SEQ ID NO:1, or by a nucleic acid having at least aboutl 85%, more preferably at least about 90% to about 95%, and most preferably at least about 99% sequence identity with the nucleic acid from SEQ ID NO:1.
In preferred embodiments, the sciellin polypeptide includes an amino terminal domain, a central domain containing comprised of sixteen repeats, and/or a carboxy terminal domain containing a LIM domain.
In preferred embodiments, the sciellin polypeptide includes a domain that includes at least one repeat, preferably 10 to 20 repeats, more preferably 14 to 18 repeats, and most preferably about 16 repeats. Generally, the domain is about 20 residues in length, and preferably, has about 70, 80, 90, or 95% sequence identity with the protein sequence shown in SEQ ID NO:2 (amino acid residues 231-543). Preferably, each repeat includes alternating stretches of hydrophobicity and hydrophilicity on hydropathy plots. Preferred hydrophobic stretches are about 3 to 10, preferably 4 to 9, most preferably, 5 to 7 residues in length. In one embodiment, the hydrophobic stretches can form beta sheets. In yet another embodiment, the invention features a sciellin polypeptide that does not include or has an inactivation in at least one repeat, preferably 10 to 20 repeats, more preferably 14 to 18 repeats, and most preferably about 16 repeats, which serves as an antagonist to one or more sciellin biological activities.
In preferred embodiments, the sciellin polypeptIide includes a LIM domain. Generally, the LIM domain is about 56 amino acids, and preferably has about 70, 80, 90, or 95% sequence identity with the protein sequence shown in SEQ ID NO:2 (amino acid residues 600-662). Preferably, the LIM domain folds in to two independent structural domains with at least one cysteine and at least one histidine residues coordinating two zinc ions. In one embodiment, the LIM domain includes at least 5, preferably 6 to 7, and most preferably 8 of the cysteins and up to 3, most preferably one histidine found in native sciellin. In one embodiment, the LIM domain has a consensus sequence CX2CX16-23HX2CX2CX2CX16-21CX2-3(C,H,D), wherein X can be any amino acid.
In a preferred embodiment, the sciellin polypeptide differs in amino acid sequence at up to 1, 2, 3, 5, or 10 residues, from a sequence in SEQ ID NO:2. In other preferred embodiments, the sciellin polypeptide differs in amino acid sequence at up to 1, 2, 3, 5, or 10% of the residues from a sequence in SEQ ID NO:2. Preferably, the differences are such that: the sciellin polypeptide exhibits a sciellin biological activity, e.g., the sciellin polypeptide retains a biological activity of a naturally occurring sciellin.
In preferred embodiments the sciellin polypeptide includes a sciellin sequence described herein as well as other N-terminal, and/or a C-terminal amino acid sequence.
In preferred embodiments, the sciellin polypeptide includes all or a fragment of an amino acid sequence from SEQ ID NO:2, fused, in reading frame, to additional amino acid residues, preferably to residues encoded by genomic DNA 5xe2x80x2 to the genomic DNA which encodes a sequence from SEQ ID NO:2.
In yet other preferred embodiments, the sciellin, polypeptide is a recombinant fusion protein having a first sciellin portion and a second pol eptide portion, e.g., a second polypeptide portion having an amino acid sequence unrelated to sciellin. The second polypeptide portion can be, e.g., any of glutathione-S-transferase, a DNA binding domain, or a polymerase activating domain. In a preferred embodiment the fusion protein can be used in a two-hybrid assay. For example, a first sciellin portion, e.g., a sciellin portion containing a LIM domain, e.g., amino acids 550 to end encoded by the last exon, can be fused to a DNA binding domain. In a two hybrid assay, the first sciellin portion is co-expressed in a cell with a second polypeptide portion containing a transcription activation domain fused to an expression library, e.g., a keratinocyte library.
In a preferred embodiment the sciellin polypeptide includes amino acid residues 231-543 of SEQ ID NO:2 In another embodiment, the sciellin polypeptide includes amino acids 600-662 of SEQ ID NO:2.
In preferred embodiments the sciellin polypeptilde has antagonistic activity, and is capable of: inhibiting formation of the cornified envelope in keratinizing tissues.
In a preferred embodiment, the sciellin polypeptide is a fragment of a naturally occurring sciellin which inhibits formation of the cornified envelope in keratinizing tissues.
Polypeptides of the invention include those which arise as a result of the existence of multiple genes, alternative transcription events, alternative RNA splicing events, and alternative translational and postranslational events. The sciellin polypeptide can be expressed in systems, e.g., cultured cells, which result in substantially the same postranslational modifications present when expressed sciellin is expressed in a native cell, or in systems which result in the omission of postranslational modifications present when expressed in a native cell.
The invention includes an immunogen which includes a sciellin polypeptide in an immunogenic preparation, the immunogen being capable of eliciting an immune response specific for the sciellin polypeptide, e.g., a humoral response, an antibody response, or a cellular response. In preferred embodiments, the immunogen comprising an antigenic determinant, e.g., a unique determinant, from a protein represented by SEQ ID NO:2.
The present invention also includes an antibody preparation specifically reactive with an epitope of the sciellin immunogen or generally of a sciellin polypeptide, preferably an epitope which consists all or in part of residues from the the amino acid sequence of SEQ ID NO:2, or an epitope, which when bound to an antibody, results in the modulation of a biological activity.
In preferred embodiments the sciellin-like polypeptide, as expressed in the cells in which it is normally expressed or in other eukaryotic cells, has a molecular weight of about 75.3 kDa as determined by SDS-PAGE.
In another embodiment, the sciellin polypeptide comprises amino acid residues 1-668 of FIG. 2 (SEQ ID NO:2).
In a preferred embodiment, the recombinant sciellin polypeptide has one or more of the following characteristics:
(i) it has the ability to form homotrimeriIc beta helices;
(ii) it acts as a precursor of the cornified envelope of keratinizing tissues;
(iii) it provides structural support to the cornified envelopes of stratum corneum cells;
(iv) it promotes adhesion between tissue elements;
(v) it promotes intracellular signalling;
(vi) it defines cell shape;
(vii) it can act as an adaptor element to promote the assembly and targeting of multiprotein complexes;
(viii) it has a molecular weight, amino acid composition or other physical characteristic of sciellin of SEQ ID NO:2;
(ix) it has an overall sequence similarity of at least 50%, preferably at least 60%, more preferably at least 70, 80, 90, or 95%, with a sciellin polypeptide of SEQ ID NO:2;
(x) it is found in human placenta;
(xi) it has a central domain composed of repeats which is preferably about 70%, 80%, 90% or 95% identical to amino acid residues 231-543 of SEQ ID NO:2;
(xii) it has a carboxyl domain containing a single LIM domain which is preferably about 70%, 80%, 90% or 95% identical to amino acid residues 600-662 of SEQ ID NO:2;
(xiii) it has a pI of about 10;
(xiv) it can be expressed in the stratum granulosum of human foreskin tissue; and
(xv) it can expressed in the peripheral cytoplasm in hair follicles, upper cell layer of epidermis, as well as the epithelium of the orali cavity, esophagus, vagina, ureter and cornea.
Also included in the invention is a composition which includes a sciellin polypeptide (or a nucleic acid which encodes it) and one or more additional components, e.g., a carrier, diluent, or solvent. The additional component can be one which renders the composition useful for in vitro and in vivo pharmaceutical or veterinary use.
In another aspect, the invention provides an isolated or substantially pure nucleic acid having or comprising a nucleotide sequence which encodes a sciellin or a sciellin polypeptide, e.g., a sciellin or a sciellin polypeptide described herein.
A preferred embodiment of the invention features a nucleic acid molecule having a nucleotide sequence at least about 85% sequence identity to a nucleotide sequence of SEQ ID NO:1. In other preferred embodiments, the sciellin polypeptide is encoded by a nucleic acid molecule having a nucleotide sequence with at least about 90% to about 95%, and more preferably about 98% to about 99% sequence identity to the nucleotide sequence from SEQ ID NO:1. In another preferred embodiment, the sciellin polypeptide is encoded by the nulceic acid molecule of SEQ ID NO:1.
In preferred embodiments, the subject sciellin nucleic acid will include a transcriptional regulatory sequence, e.g. at least one of a transcriptional promoter or transcriptional enhancer sequence, operably linked to the sciellin gene sequence (also referred to as LAMG3), e.g., to render the sciellin gene sequence suitable for use as an expression vector.
In yet a further preferred embodiment, the nucleic acid which encodes a sciellin polypeptide of the invention, hybridizes under stringent conditions to a nucleic acid probe corresponding to at least 12 consecutive nucleotides ofiSEQ ID NO:1. More preferably, the nucleic acid probe corresponds to at least 20 consecutive nucleotides from SEQ ID NO:1.
The invention also provides a probe or primer which includes or comprises a substantially purified oligonucleotide. The oligonucleotide includes a region of nucleotide sequence which hybridizes under stringent conditions to at least 10 consecutive nucleotides of sense or antisense sequence from SEQ ID NO:1, or naturally occurring mutants thereof. In preferred embodiments, the probe or primer further includes a label group attached thereto. The label group can be, e.g., a radioisotope, a fluorescen t compound, an enzyme, and/or an enzyme co-factor. Preferably the oligonucleotide is at least 10 and less than 20, 30, 50, 100, or 150 nucleotides in length.
The invention involves nucleic acids, e.g., RNA or DNA, encoding a sciellin polypeptide of the invention. This includes double stranded nucleic acids as well as coding and antisense single strands.
In another aspect, the invention features a cell or purified preparation of cells which include a sciellin transgene, or which otherwise misexpress a sciellin gene. The cell preparation can consist of human or non human cells, e.g., rodent cells, e.g., mouse or rat cells, rabbit cells, or pig cells. In preferred embodiments, the cell or cells include a sciellin transgene, e.g., a heterologous form of a sciellin gene, e.g., a gene derived from humans (in the case of a non-human cell). The sciellin transgene can be misexpressed, e.g., overexpressed or underexpressed. In other preferred embodiments, the cell or cells include a gene which misexpress an endogenous sciellin gene, e.g., a gene the expression of which is disrupted, e.g., a knockout. Such cells can serve as a model for studying disorders which are related to mutated or mis-expressed sciellin alleles or for use in drug screening.
In another aspect, the invention features a transgenic sciellin animal, e.g., a rodent, e.g., a mouse or a rat, a rabbit, a pig, a goat, or a cow. In preferred embodiments, the transgenic animal includes (and preferably express) a heterologous form of a sciellin gene, e.g., a gene derived from humans. In a further embodiment, the sciellin transgene includes a tissue specific promoter, e.g., a K14 promoter. In other preferred embodiments, the animal has an endogenous sciellin gene which is misexpressed, e.g., a knockout. Such a transgenic animal can serve as a model for studying disorders which are related to mutated or mis-expressed sciellin alleles or for use in drug screening.
In another aspect, the invention features, a method of inducing differentiation of keratinocytes. The method includes contacting a keratiniocyte with an amount of a sciellin molecule described herein, or a sciellin agent, sufficientito induce differentiation. The sciellin agent can be an agonist or an antagonist of sciellin activity. The method can be performed in vivo, or in vitro. In in vivo methods the sciellin is administered to the subject. The administration can be directed to the site where differentiation is desired, e.g., by topical application or by injection, or administered in a systemic fashion.
In another aspect, the invention features, a method of promoting hair growth. The method includes contacting a hair stem cells with an amount of a sciellin molecule described herein or a sciellin agent, sufficient to induce hair growth. The sciellin agent can be an agonist or an antagonist of sciellin activity. The method can be performed in vivo, or in vitro. In in vivo methods the sciellin is administered to the subject. The administration can be directed to the site where hair growth is desired, e.g., by topical application or by injection, or administered in a systemic fashion.
In another aspect, the invention features, a method of promoting adhesion of a first tissue element to a second tissue element. The method includes contacting one or both of the first tissue element and the second tissue element with an amount of a sciellin molecule described herein or a sciellin agent, sufficient to promote adhesion. The sciellin agent can be an agonist or an antagonist of sciellin activity. The method can be performed in vivo, or in vitro. In in vivo methods the sciellin is administered to the subject. The administration can be directed to the site where adhesion is desired, e.g., by topical application or by injection, or administered in a systemic fashion.
A tissue element can be a cell or a multi-cellularion acellular structure. Examples of tissue elements include, skin cells, e.g., epidermal or dermal cells, or e.g., foreskin tissue, e.g., stratum granulosum, basement membrane or components of the basement membrane, hair follicles, epithelium in the oral cavity, esophagus, vagina, ureter and cornea, or any cell or structure which in normal, non-traumatized, or non-diseased tissue is adjascent or adhered to a specific tissue element recited herein.
In preferred embodiments the molecule is exogenous (e.g., administered to a subject) or is recombinant.
In preferred embodiments the method is an vivo method. In vivo methods can be autologous, allogeneic, or xenogeneic. In autologous methods, adhesion between two tissue elements from the subject is promoted. In allogeneic methods, adhesion between a recipient tissue element and a donor tissue element from an allogeneic donor is promoted. In xenogeneic methods, adhesion between a recipient tissue element and a donor tissue element from a xenogeneic donor is promoted. Thus, one element can be a donor tissue element which is implanted into a recipient subject.
In preferred embodiments the first tissue is healthy tissue, e.g., skin tissue, and the second tissue is wounded, e.g., burned, diseased, traumatized, cut, and the tissue, or is a wound bed. For example, the first tissue is skin tissue, from the subject or from a donor, and the second tissue is wounded, e.g., burned or abraided tissue.
In preferred embodiments the first tissue and second tissue element are normally adhered but have become detached from one another due to trauma, burn or other physical injury, disease, or age.
In preferred embodiments: the first tissue element is a dermal cell and the second tissue element is an epidermal cell; the first tissue element is e.g., foreskin tissue, e.g., stratum granulosum, hair follicles, part of the epithelium in the oral cavity, esophagus, vagina, ureter and cornea and the second tissue element is a cell or structure which in normal, non-traumatized, or non-diseased tissue is adjacent or adhered to said epithelium.
The administration of sciellin can be repeated.
In another aspect, the invention features a method of promoting wound healing in a subject. The method includes administering an amount of a sciellin molecule described herein or a sciellin agent, sufficient to promote healing to the wound. The sciellin agent can be an agonist or an antagonist of sciellin activity. The administration can be directed to the site where healing is desired, e.g., by topical application or by injection, or administered in a systemic fashion.
The wound can be in any tissue, but preferably in a tissue in which the sciellin normally occurs. Examples skin, e.g., foreskin tissue, e.g., stratum granulosum, hair follicles, tissues of the eye, e.g., the cornea, the basement membrane the epithelium in, e.g., the oral cavity, esophagus, vagina, ureter, or any tissue which in normal, non-traumatized, or non-diseased tissue is adjacent or adhered thereto.
In preferred embodiments the molecule is exogenous (e.g., administered to a subject) or is recombinant.
In preferred embodiments the wound tissue is burned, diseased, traumatized, cut, the subject of immune attack, e.g., autoimmune attack, or abraided.
The administration of sciellin can be repeated.
In another aspect, the invention provides, a method of treating or preventing in a subject a sciellin- related disorder. The method includes: administering to the subject an effective amount of sciellin molecule, or a sciellin agonist, effective to treat or prevent the sciellin- related disorder in the subject. The sciellin agent can be an agonist or an antagonist of sciellin activity. The administration can be directed to the site where treatment or prevention is desired, e.g., by topical application or by injection, or administered in a systemic fashion.
In preferred embodiments, the subject is a mammal, e.g., human or non-human.
Such sciellin-related disorders include, e.g., a disorder associated with the misexpression of sciellin; a skin disorder, e.g., a foreskin disorder; an epidermal disorder, e.g., a disorder characterized by disturbed epidermal characterization or a disorder in the stratum granulosum, e.g., ichthyosis; an ectodermal disorder, e.g., ectodermal dysplasia; a dermal disorder; a hair growth disorder, e.g., congenital allopecia; a disorder associated with a genetic lesion at chromosome 13, region q22; a disorder associated with abnormal levels, e.g., abnormally low levels, of adhesion between tissues; a disorder associated with the basement membrane; a disorder associated with abnormal keratinocyte activity; e.g., abnormal activity of keratinocytes in the epithelium of oral cavity, esophagus, vagina, ureter or cornea; or an inflammatory condition.
In preferred embodiments the molecule is exogenous (e.g., administered to a subject) or is recombinant.
The administration of sciellin can be repeated.
In another aspect, the invention provides, a method of determining if a subject is at risk for a disorder related to a lesion in or the misexpression of a gene which encodes a sciellin described herein.
Such disorders include, e.g., a disorder associated with the misexpression of sciellin; a skin disorder, e.g., a foreskin disorder; an epidermal disorder, e.g., a disorder characterized by disturbed epidermal characterization or a disorder in the stratum granulosum, e.g., ichthyosis; an ectodermal disorder, e.g., ectodermal dysplasia; a dermal disorder; a hair growth disorder, e.g., congenital allopecia; a disorder associated with a genetic lesion at chromosome 13, region q22; a disorder associated with abnormal levels, e.g., abnormally low levels, of adhesion between tissues; a disorder associated with the basement membrane; a disorder associated with abnormal keratinocyte activity; e.g., abnormal activity of keratinocytes in the epithelium of oral cavity, esophagus, vagina, ureter or cornea; or an inflammatory condition.
The method includes one or more of the following:
detecting, in a tissue of the subject, the presencelor absence of a mutation which affects the expression of the sciellin gene, or other gene which encodes a subunit of sciellin, e.g., detecting the presence or absence of a mutation in a region which controls the expression of the gene, e.g., a mutation in the 5xe2x80x2 control region;
detecting, in a tissue of the subject, the presence or absence of a mutation which alters the structure of the sciellin gene;
detecting, in a tissue of the subject, the misexpression of the sciellin gene, at the mRNA level, e.g., detecting a non-wild type level of a sciellin mRNA;
detecting, in a tissue of the subject, the misexpression of the sciellin gene, at the protein level, e.g., detecting a non-wild type level of a sciellin polypeptide.
In preferred embodiments the method includes: ascertaining the existence of at least one of: a deletion of one or more nucleotides from the sciellin gene; an insertion of one or more nucleotides into the gene, a point mutation, e.g., a substitution of one or more nucleotides of the gene, a gross chromosomal rearrangement of the gene, e.g., a translocation, inversion, or deletion.
For example, detecting the genetic lesion can include: (i) providing a probe/primer including an oligonucleotide containing a region of nucleotide sequence which hybridizes to a sense or antisense sequence from SEQ ID NO:1, or naturally occurring mutants thereof or 5xe2x80x2 or 3xe2x80x2 flanking sequences naturally associated with the LAMG3 gene; (ii) exposing the probe/primer to nucleic acid of the tissue; and detecting, by hybridization, e.g., in situ hybridization, of the probe/primer to the nucleic acid, the presence or absence of the genetic lesion.
In preferred embodiments detecting the misexpression includes ascertaining the existence of at least one of: an alteration in the level of a messenger RNA transcript of the sciellin gene; the presence of a non-wild type splicing pattern of a messenger RNA transcript of the sciellin gene; or a non-wild type level of sciellin.
Methods of the invention can be used prenatally or to determine if a subject""s offspring will be at risk for a disorder.
In preferred embodiments the method includes determining the structure of a sciellin gene, an abnormal structure being indicative of risk for the disorder.
In preferred embodiments the method includes contacting a sample form the subject with an antibody to the sciellin protein or a nucleic acid which hybridizes specifically with the sciellin gene,.
In another aspect, the invention features, a method of evaluating a compound for the ability to interact with, e.g., bind, a subject sciellin polypeptide, e.g., sciellin or a fragment thereof, e.g., a central repeat domain, or a LIM domain, of sciellin. The method includes: contacting the compound with the subject sciellin polypeptide; and evaluating ability of the compound to interact with, e.g., to bind or form a complex with the subject sciellin polypeptide. This method can be performed in vitro, e.g., in a cell free system, or in vivo, e.g., in a two-hybrid interaction trap assay. This method can be used to identify naturally occurring molecules which interact with subject sciellin polypeptide. It can also be used to find natural or synthetic inhibitors of subject sciellin polypeptide.
In another aspect, the invention features, a method of evaluating a compound, e.g., a polypeptide, e.g., a naturally occurring ligand of or a naturally occuring substrate to which binds a subject sciellin polypeptide, e.g., sciellin or a fragment thereof, e.g., a central repeat domain, or a LIM domain, of sciellin, for the ability to bind a subject sciellin polypeptide. The method includes: contacting the compound with the subject sciellin polypeptide; and evaluating the ability of the compound to interact with, e.g., to bind or form a complex with the subject sciellin polypeptide, e.g., the ability of the compound to inhibit a subject sciellin polypeptide/ligand interaction. This method can be performed in vitro, e.g., in a cell free system, or in vivo, e.g., in a two-hybrid interaction trap assay. This method can be used to identify compounds, e.g., fragments or analogs of a subject sciellin polypeptide, which are agonists or antagonists of a subject sciellin polypeptide.
In another aspect, the invention features, a method of evaluating a first compound, e.g., a subject sciellin polypeptide, e.g., sciellin or a fragment thereof, e.g., a central repeat domain, or a LIM domain, of sciellin, for the ability to bind a second compound, e.g., a second polypeptide, e.g., a naturally occurring ligand of or substrate to which binds a subject sciellin polypeptide. The method includes: contacting the first compound with the second compound; and evaluating the ability of the first compound to form a complex with the second compound. This method can be performed in vitro, e.g., in a cell free system, or in vivo, e.g., in a two-hybrid interaction trap assay. This method can be used to identify compounds, e.g., fragments or analogs of a subject sciellin polypeptide, which are agonists or antagonists of a subject sciellin polypeptide.
In yet another aspect, the invention features a method for evaluating a compound, e.g., for the ability to modulate an interaction, e.g., the ability to inhibit an interaction of a subject sciellin polypeptide, e.g., sciellin or a fragment thereof, e.g., a central repeat domain, or a LIM domain, of sciellin, with a second polypeptide, e.g., a polypeptide, e.g., a natural ligand of the of or a substrate wo which binds a subject sciellin polypeptide, or a fragment thereof. The method includes the steps of (i) combining the second polypeptide (or preferably a purified preparation thereof), a subject sciellin polypepltide, (or preferably a purified preparation thereof), and a compound, e.g., under conditions wherein in the absence of the compound, the second polypeptide, and the subject sciellin polypeptide, are able to interact, e.g., to bind or form a complex; and (ii) detecting the interaction, e.g., detecting the formation (or dissolution) of a complex which includes the second polypeptide, and the subject sciellin polypeptide. A change, e.g., a decrease or increase, in the formation of the complex in the presence of a compound (relative to what is seen in the absence of the compound) is indicative of a modulation, e.g., an inhibition or promotion, of the interaction between the second polypeptide, and the subject sciellin polypeptide. In preferred embodiments: the second polypeptide, and the subject sciellin polypeptide, are combined in a cell-free system and contacted with the compound; the cell-free system is selected from a group consisting of a cell lysate and a reconstituted protein mixture; the subject sciellin polypeptide, and the second polypeptide are simultaneously expressed in a cell, and the cell is contacted with the compound, e.g. in an interaction trap assay (e.g., a two-hybrid assay).
In yet another aspect, the invention features a two-phase method (e.g., a method having an in vitro, e.g., in a cell free system, and an in vivo phase) for evaluating a compound, e.g., for the ability to modulate, e.g., to inhibit or promote, an interaction of a subject sciellin polypeptide subject sciellin polypeptide ,e.g., sciellin or a fragment thereof, e.g., a central repeat domain, or a LIM domain, of sciellin, with a second compound, e.g., a second polypeptide, e.g., a naturally occurring ligand of or a substrate to which binds a subject sciellin polypeptide, or a fragment thereof. The method includes steps (i) and (ii) of the method described immediately above performed in vitro, and further includes: (iii) determining if the compound modulates the interaction in vitro, e.g., in a cell free system, and if so; (iv) administering the compound to a cell or animal; and (v) evaluating the in vivo effect of the compound on an interaction, e.g., inhibition, of a subject sciellin polypeptide, with a second polypeptide.
In another aspect, the invention features, a method of evaluating a compound for the ability to bind a nucleic acid encoding a subject sciellin polypeptide, e.g., sciellin or a fragment thereof, e.g., a central repeat domain or a LIM domain. The method includes: contacting the compound with the nucleic acid; and evaluating ability of the compound to form a complex with the nucleic acid.
In another aspect, the invention features a method of making a sciellin polypeptide, e.g., a peptide having a non-wild type activity, e.g., an antagonist, agonist, or super agonist of a naturally occurring sciellin polypeptide, e.g., a naturally occurring sciellin polypeptide. The method includes: altering the sequence of a sciellin polypeptide, e.g., altering the sequence , e.g., by substitution or deletion of one or more residues of a non-conserved region, a domain or residue disclosed herein, and testing the altered polypeptide for the desired activity.
In another aspect, the invention features a method of making a fragment or analog of a sciellin polypeptide having a biological activity of a naturally occurring sciellin polypeptide. The method includes: altering the sequence, e.g., by substitution or deletion of one or more residues, of a sciellin polypeptide, e.g., altering the sequence of a non-conserved region, or a domain or residue described herein, and testing the altered polypeptide for the desired activity.
In another aspect, the invention features, a human cell, e.g., a skin cell, e.g., an epithelial cell, e.g., a keratinocyte, transformed with nucleic acid which encodes a subject sciellin polypeptide.
In another aspect, the invention includes: a sciellin nucleic acid, e.g., a sciellin nucleic acid inserted into a vector; a cell transformed with a sciellin nucleic acid; a sciellin made by culturing a cell transformed with a sciellin nucleic acid; and a method of making a sciellin polypeptide including culturing a cell transformed with a sciellin nucleic acid.
A xe2x80x9cheterologous promoterxe2x80x9d, as used herein is a promoter which is not naturally associated with a gene or a purified nucleic acid.
A xe2x80x9cpurifiedxe2x80x9d or xe2x80x9csubstantially purexe2x80x9d or isolated xe2x80x9cpreparationxe2x80x9d of a polypeptide, as used herein, means a polypeptide that has been separated from other proteins, lipids, and nucleic acids with which it naturally occurs. Preferably, the polypeptide is also separated from substances, e.g., antibodies or gel matrix, e.g., polyacrylamide, which are used to purify it. Preferably, the polypeptide constitutes at least 10, 20, 5p 70, 80 or 95% dry weight of the purified preparation. Preferably, the preparation contains: sufficient polypeptide to allow protein sequencing; at least 1, 10, or 100 xcexcg of the polypeptide; at least 1, 10, or 100 mg of the polypeptide.
A xe2x80x9cpurified preparation of cellsxe2x80x9d, as used herein, refers to, in the case of plant or animal cells, an in vitro preparation of cells and not an entire intact plant or animal. In the case of cultured cells or microbial cells, it consists of a preparation of at least 10% and more preferably 50% of the subject cells.
A xe2x80x9ctreatmentxe2x80x9d, as used herein, includes any therapeutic treatment, e.g., the administration of a therapeutic agent or substance, e.g., a drug.
As used herein, the term xe2x80x9csubjectxe2x80x9d refers to human and non-human animals. In preferred embodiments, the subject is a human, e.g., person, e.g., a person having a sciellin related disorder. The term xe2x80x9cnon-human animalsxe2x80x9d of the, invention includes all vertebrates, e.g., mammals and non-mammals, such as non-human primates, ruminants, birds, amphibians, reptiles.
An xe2x80x9cisolatedxe2x80x9d or xe2x80x9cpure nucleic acidxe2x80x9d, e.g., a substantially pure DNA, is a nucleic acid which is one or both of: not immediately contiguous with either one or both of the sequences, e.g., coding sequences, with which it is immediately contiguous (i.e., one at the 5xe2x80x2 end and one at the 3xe2x80x2 end) in the naturally-occurring genome of the organism from which the nucleic acid is derived; or which is substantially free of a nucleic acid sequence with which it occurs in the organism from which the nucleic acid is derived. The term includes, for example, a recombinant DNA which is incorporated into a vector, e.g., into an autonomously replicating plasmid or virus, or into the genomic DNA of a prokaryote or eukaryote, or which exists as a separate molecule (e.g., a cDNA or a genomic DNA fragment produced by PCR or restriction endonuclease treatment) independent of other DNA sequences. Substantially pure DNA can also includes a recombinant DNA which is part of a hybrid gene encoding sequence.
xe2x80x9cSequence identity or homologyxe2x80x9d, as used herein, refers to the sequence similarity between two polypeptide molecules or between two nucleic acid molecules. When a position in both of the two compared sequences is occupied by the same base or amino acid monomer subunit, e.g., if a position in each of two DNA molecules is occupied by adenine, then the molecules are homologous or sequence identical at that position. The percent of homology or sequence identity between two sequences is a function of the number of matching or homologous identical positions shared by the sequences divided by the number of positions comparedxc3x97100. For example, if 6 of 10, of the positions in two sequences are the same then the two sequences are 60% homologous or have 60% sequence identity. By way of example, the DNA sequences ATTGCC and TATGGC share 50% homology or sequence identity. Generally, a comparison is made when two sequences are aligned to give maximum homology. Unless otherwise specified xe2x80x9cloop out regionsxe2x80x9d, e.g., those arising from, from deletions or insertions in one of the sequences are counted as mismatches.
The comparison of sequences and determination of percent homology between two sequences can be accomplished using a mathematical algorithim. Preferably, the alignment can be performed using the Clustal Method. Multiple alignment paramethers include GAP Penalty=10, Gap Length Pehalty=10. For DNA alignments, the pairwise alignment paramenters can beg Htuple=2, Gap penalty=5, Window=4, and Diagonal saved=4. For protein alignments, the pairwise alignment parameters can be Ktuple=1, Gap penalty=3, Window=5, and Diagonals Saved=5.
Additional non-limiting example of a mathematical algorithim utilized for the comparison of sequences is the algorithm of Karlin and Altschul (1990) Proc. Natl. Acad. Sci. USA 87:2264-68, modified as in Karlin and Altschul (1993) Proc. Natl. Acad. Sci. USA 90:5873-77. Such an algorithm is incorporated into the NBLAST and XBLAST programs (version 2.0) of Altschul, et al. (1990) J. Mol. Biol. 215:403-10. BLAST nucleotide searches can be performed performed with the NBLAST program, score=100, wordlength=12 to obtain nucleotide sequences homologous to nucleic acid molecules of the invention. BLAST protein searches can be performed with the XBLAST program, score=50, wordlength=3 to obtain amino acid sequences homologous to protein molecules of the invention. To obtain gapped alignments for comparison purposes, Gapped BLAST can be utilized as described in Altschul et al., (1997) Nucleic Acids Research 25(17):3389-3402. When utilizing BLAST and Gapped BLAST programs, the default parameters of the respective programs (e.g., XBLAST and NBLAST) can be used. See http://www ncbi.nlm.nih.gov. Another preferred, non-limiting example of a mathematical algorithim utilized for the comparison of sequences is the algorithm of Myers and Miller, CABIOS (1 989). Such an algorithm is incorporated into the ALIGN program (version 2.0) which is part of the GCG sequence alignment software package. When utilizing the ALIGN program for comparing amino acid sequences, a PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4 can be used.
The terms xe2x80x9cpeptidesxe2x80x9d, xe2x80x9cproteinsxe2x80x9d, and xe2x80x9cpolypeptidesxe2x80x9d are used interchangeably herein.
As used herein, the term xe2x80x9ctransgenexe2x80x9d means a nucleic acid sequence (encoding, e.g., one or more subject sciellin polypeptides), which is partly or entirely heterologous, i.e., foreign, to the transgenic animal or cell into which it is introduced, or, is homologous to an endogenous gene of the transgenic animal or cell into which it is introduced, but which is designed to be inserted, or is inserted, into the animal""s genome in such a way as to alter the genome of the cell into which it is inserted (e.g., it is inserted at a location which differs from that of the natural gene or its insertion results in a knockout). A transgene can include one or more transcriptional regulatory sequences and any other nucleic acid, such as introns, that may be necessary for optimal expression of the selected nucleic acid, all operably linked to the selected nucleic acid, and may include an enhancer sequence.
As used herein, the term xe2x80x9ctransgenic cellxe2x80x9d refersito a cell containing a transgene.
As used herein, a xe2x80x9ctransgenic animalxe2x80x9d is any animal in which one or more, and preferably essentially all, of the cells of the animal includes a transgene. The transgene can be introduced into the cell, directly or indirectly by introduction into a precursor of the cell, by way of deliberate genetic manipulation, such as by microinjection or by infection with a recombinant virus. This molecule may be integrated within a chromosome, or it may be extrachromosomally replicating DNA.
As used herein, the term xe2x80x9ctissue-specific promoterxe2x80x9d means a DNA sequence that serves as a promoter, i.e., regulates expression of a selected DNA sequence operably linked to the promoter, and which effects expression of the selected DNA sequence in specific cells of a tissue, such as mammary tissue. The term also covers so-called xe2x80x9cleakyxe2x80x9d promoters, which regulate expression of a selected DNA primarily in one tissue, but cause expression in other tissues as well.
xe2x80x9cUnrelated to a sciellin amino acid or nucleic acid sequencexe2x80x9d means having less than 30% sequence identity, less than 20% sequence identity, or, preferably, less than 10% homology with a naturally occuring sciellin sequence disclosed herein.
A polypeptide has sciellin biological activity if it has one or more of the properties of sciellin disclosed herein. A polypeptide has biological activity if it is an antagonist, agonist, or super-agonist of a polypeptide having one of the properties of sciellin disclosed herein.
xe2x80x9cMisexpressionxe2x80x9d, as used herein, refers to a non-wild type pattern of gene expression, at the RNA or protein level. It includes: expression at non-wild type levels, i.e., over or under expression; a pattern of expression that differs from wild type in terms of the time or stage at which the gene is expressed, e.g., increased or decreased expression (as compared with wild type) at a predetermined developmental period or stage; a pattern of expression that differs from wild type in terms of decreased expression (as compared with wild type) in a predetermined cell type or tissue type; a pattern of expression that differs from wild type in terms of the splicing size, amino acid sequence, post-transitional modification, or biological activity of the expressed polypeptide; a pattern of expression that differs from wild type in terms of the effect of an environmental stimulus or extracellular stimulus on expression of the gene, e.g., a pattern of increased or decreased expression (as compared with wild type) in the presence of an increase or decrease in the strength of the stimulus.
Subject, as used herein, can refer to a mammal, e.g., a human, or to an experimental or animal or disease model. The subject can also be a non-human animal, e.g., a horse, cow, goat, or other domestic animal.
As described herein, one aspect of the invention features a substantially pure (or recombinant) nucleic acid which includes a nucleotide sequence encoding a sciellin polypeptide and/or equivalents of such nucleic acids. The term nucleic acid as used herein can include fragments and equivalents. The term equivalent refers to nucleotide sequences encoding functionally equivalent polypeptides. Equivalent nucleotide sequences will include sequences that differ by one or more nucleotide substitutions, additions or deletions, such as allelic variants, and include sequences that differ from the nucleotide sequences disclosed herein by degeneracy of the genetic code.
The practice of the present invention will employ, unless otherwise indicated, conventional techniques of cell biology, cell culture, molecular biology, transgenic biology, microbiology, recombinant DNA, and immunology, which are within the skill of the art. Such techniques are described in the literature. See, for example, Molecular Cloning A Laboratory Manual, 2nd Ed., ed. by Sambrook, Fritsch and Maniatis (Cold Spring Harbor Laboratory Press: 1989); DNA Cloning, Volumes I and II (D. N. Glover ed., 1985); Oligonucleotide Synthesis (M. J. Gait ed., 1984); Mullis et al. U.S. Pat. No: 4,683,195; Nucleic Acid Hybridization (B. D. Hames and S. J. Higgins eds. 1984); Transcription And Translation (B. D. Hames and S. J. Higgins eds. 1984); Culture Of Animal Cells (R. I. Freshney, Alan R. Liss, Inc., 1987); Immobilized Cells And Enzymes (IRL Press, 1986); B. Perbal, A Practical Guide To Molecular Cloning (1984); the treatise, Methods In Enzymology (Academic Press, Inc., N.Y.); Gene Transfer Vectors For Mammalian Cells (J. H. Miller and M. P. Calos eds., 1987, Cold Spring Harbor Laboratory); Methods In Enzymology, Vols. 154 and 155 (Wu et al. eds.), Immunochemical Methods In Cell And Molecular Biology (Mayer and Walker, eds., Academic Press, London, 1987); Handbook Of Experimental Immunology, Volumes I-IV (D. M. Weir and C. C. Blackwell, eds., 1986); Manipulating the Mouse Embryo, (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1986).
High stringency aqueous hybridization of nucleic acids can be conducted at 68xc2x0 C., in 6X SSC, 0.5% SDS, followed by a wash in 2X SSC, 0.1% SDS at room temperature; a wash in 0.1X SSC, 0.5% SDS at 37xc2x0 C.; and a final wash in 0.1 X SSC, 0.5% SDS at 68xc2x0 C. (Molecular Cloning, A Laboratory Manual, 2d Ed., ed. by Sambrook, Fritsch and Maniatis, Cold Spring Harbor Laboratory Press, 1989, 9.52-9.55).
Other features and advantages of the inventions will be apparent from the following detailed description, and from the claims.